Small chemical moieties, such as heavy metal ions, can and often do affect the environment and biological systems. These effects become astounding when it is realized that minute quantities of these small moieties are involved. Moreover, the presence or absence of low concentrations of small moieties in the environment can have long term consequences. Minute quantities of metallic cations, such as mercury cations, can regulate, influence, change or toxify the environment or biological systems.
The detection, removal, addition or neutralization of such minute quantities constitutes a focal point for continued research in many fields. For example, many efforts have been made to detect and remove minute, toxic amounts of heavy metal ions such as cadmium or mercury from the environment. The efforts often have not been successful or economical for widespread application. On the other hand, minute concentrations of other heavy metals are important for the proper function of biological organisms. Zinc, for example, plays a major role in wound healing. The function of magnesium in plant photosynthesis is another.
Heavy metal can exhibit dual roles. Mercury is used in diuretics, topical anti-bacterial agents, skin antiseptics, ointments, and in chemical manufacturing operations. Yet when ingested by mammals, such as from drinking water, it is highly toxic in very small amounts. Hence, detection and quantification of minute concentrations of heavy metals in drinking water and other media would serve exploratory, safety and regulatory goals.
It would, therefore, be highly desirable to identify and control minute quantities of heavy metals, e.g., mercury cations, in aqueous biological or inanimate systems. In most contexts, however, the detection, removal, addition or neutralization of heavy metals, is a difficult and expensive and often unfeasible if not impossible task. Other metallic contaminants often mimic the heavy metal of interest. Measurement interference will result. Moreover, the detection methods employed today are usually not sufficiently sensitive at the minute quantities under consideration. Consequently, it is desirable to develop reliable and economic methods for accurately identifying and controlling minute quantities of heavy metals in aqueous systems.
Antibodies would seem to be uniquely suited for this task. Their high degree of specificity for a known antigen would avoid the interference caused by contaminants. The sensitivity of antibodies in the picomolar or lower range would permit accurate and efficient targeting and detection of such minute levels.
Monoclonal antibodies, of course, come to mind as especially suited agents for practice of this technique. Since Kohler and Milstein published their article on the use of somatic cell hybridization to produce monoclonal antibodies (Nature 256:495 (1974)), immunologists have developed many monoclonal antibodies which strongly and specifically immunoreact with antigens.
Notwithstanding this suggestion, the conventional understanding about immunology teaches that antibodies against small moieties, such as heavy metals, cannot be developed. The mammal immunization step, which is key for the production of monoclonal antibodies, typically requires a molecule that is large enough to cause antigenic reaction. Medium sized molecules (haptens), which are not of themselves immunogenic, can induce immune reaction by binding to an immunogenic carrier. Nevertheless, immunologists view small moieties such as metallic cations, as not large or structurally complex enough to elicit an antibody response. One theory appears to hold that electron rich rings such as those associated with benzene and carbohydrates are needed at a minimum to cause immunogenicity. V. Butler, S. Beiser, Adv. Immunol., 17, 255 (1973). The molecular size and lack of complexity of an inorganic moiety is thought to render it insufficient for eliciting an antibody response. To date, therefore, no monoclonal antibodies which immunoreact with mercury cations per se have been reported in the literature.
Several immunologists have reported production of monoclonal antibodies to metallic ion chelates. For. example, in U.S. Pat. No. 4,722,892, monoclonal antibodies are disclosed which immunoreact with a complex of a chelating agent, such as ethylene diamine tetracetate (EDTA), and a heavy metal such as indium. In EPO Patent Application 0235457, monoclonal antibodies that immunoreact with a chelate of gold cyanate and carbonate coating are disclosed. In these instances, however, the monoclonal antibodies bind with the metal chelate complex rather than the bare metallic ion itself. Disadvantages of these methods include: the complicated reagents involved in detection, lack of simple tests that discriminate among antigens, cross-reactivity with chelates of other antigens and cross-reactivity with the chelate itself.
Other instances of monoclonal antibody combinations with metals involve metal tags. The metal chelates are bound to the antibody at a site remote from the antigen binding site or sites. The metal or metal chelate is not the antigen. Instead, it is a tag to indicate the presence of the monoclonal antibody when it reacts with its specific antigen. See for example, V. P. Torchilian et al., Hybridoma, 6, 229 (1987); and C. F. Meares, Nuclear Medical Biology, 13, 311-318 (1986).
It is therefore, an object of the invention to develop polypeptides that immunoreact with heavy metals per se and with mercury ions in particular. It is another object of the invention to develop methods for detecting or neutralizing heavy metals within, adding heavy metals to, or removing heavy metals from biological or inanimate systems through the use of the monoclonal antibodies. Further objects include the development of nucleic acid sequences coding for polypeptides which immunoreact with mercury cations and the development of methods of expressing these nucleic acid sequences to produce metal binding polypeptides.